Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.
Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.
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DOI：
10.1152/ajplung.00267.2016
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发表时间：
2017-05
期刊：
影响因子：
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通讯作者：
H. White;Yi Jin;L. Chicoine;Bernadette Chen;Yusen Liu;L. Nelin
中科院分区：
文献类型：
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作者：
H. White;Yi Jin;L. Chicoine;Bernadette Chen;Yusen Liu;L. Nelin
我们先前已经证明，人肺微血管内皮细胞(HPMVECs)的低氧增殖依赖于表皮生长因子受体(EGFR)的激活。为了确定导致增殖的下游信号，我们验证了这样的假设，即低氧诱导的hPMVECs增殖需要EGFR介导的细胞外信号调节激酶(ERK)的激活，从而诱导精氨酸酶II的诱导。为了验证这一假设，将hPMVEC培养在常氧(21%O2，5%CO2)和低氧(1%O2，5%CO2)中，并进行EGFR、精氨酸酶II、磷酸化ERK(PERK)和总ERK(ERK)的Western blotting。缺氧导致hPMVECs中EGFR、PERK和精氨酸酶II蛋白水平高于常氧。为了研究EGFR在缺氧诱导的这些变化中的作用，将针对EGFR的siRNA或干扰的siRNA导入hPMVEC，并将其置于缺氧中。使用siRNA抑制EGFR可以减少低氧诱导的PERK和精氨酸酶II的表达以及低氧诱导的活细胞数量的增加。然后用赋形剂、EGFR抑制剂(AG1478)或ERK途径抑制剂(U0126)处理hPMVEC，并将其置于低氧中。药物抑制EGFR可显著减轻缺氧引起的PERK水平升高。AG1478和U0126也能显著抑制缺氧诱导的hPMVECs活性增加。将含精氨酸酶II的腺病毒载体(AdArg2)导入人脐静脉内皮细胞，过表达精氨酸酶II可挽救U0126介导的缺氧hPMVECs活细胞数的减少。我们的发现表明，低氧激活EGFR导致ERK的磷酸化，这是低氧诱导精氨酸酶II和细胞增殖所必需的。
We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.